In many animal cells, stimulation of cell surface receptors coupled to G proteins or tyrosine kinases mobilizes Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. The ensuing Ca2+ entry regulates a wide variety of effector cell responses including transcription, motility, and proliferation. CRAC channels exhibit a unique biophysical fingerprint characterized by exquisite Ca2+-selectivity, store-operated gating, and distinct pore properties, and serve as fascinating ion channels for understanding the biophysical mechanisms of ion permeation and gating. Moreover, because CRAC channels sit squarely at the nexus of the cellular Ca2+ signaling network in many cells, aberrant CRAC channel function is implicated in the etiology of several diseases including chronic inflammation, muscle weakness, and a severe combined immunodeficiency syndrome. Much has been learned in the last decade of the physiological roles of CRAC channels in different tissues and the cellular choreography of the CRAC channel activation process. Still, the molecular and structural basis of how depletion of ER Ca2+ stores regulates the opening of the CRAC channel pore continues to remain poorly understood. Opening of CRAC channels is governed through direct interactions between the pore-forming Orai proteins, and the ER Ca2+ sensor, STIM1, but how STIM1 binding transduces opening of the Orai1 channel pore remains unclear. Here, we propose a multi-pronged approach that will investigate several mechanistic aspects of the CRAC channel gating process, focusing on the molecular rearrangements in the CRAC channel pore and the conformational alterations in STIM1. We will employ an interdisciplinary experimental design for these studies that combines patch-clamp electrophysiology, cysteine mutagenesis, FRET microscopy, biochemical cross-linking, and protein engineering. Using the Orai1 protein as a prototypic CRAC channel, our goals are to: (1) test the hypothesis that opening of the CRAC channel pore occurs through rotation of the TM1 helix, thereby reorienting the hydrophobic V102 side-chains to remove an energy barrier, (2) elucidate the inner pore architecture and its role in regulating ion conduction in Orai1 channels, and (3) examine the conformational rearrangements in the critically important cytoplasmic region of STIM1 during activation and binding to Orai1. These studies will significantly advance our understanding of the molecular and structural mechanisms of CRAC channel activation and open new avenues for harnessing the therapeutic potential of CRAC channels for disease intervention.